Progress in optical microscopy has led to the emergence of a wide range of systems for fluorescence imaging of biological samples. For example, confocal [“Microscopy apparatus”, U.S. Pat. No. 3,013,467 to Minsky] and two-photon [Denk et al., “Two-Photon Laser Scanning Fluorescence Microscopy”, Science vol. 48, p. 73-76 (1990)] laser scanning fluorescence microscopes having better spatial resolution than conventional wide-field microscopy are now commonly employed for imaging narrow sections of biological structures, in which features of interest are tagged with fluorescent markers. Both confocal and two-photon laser microscopes can provide depths of field of the order of only a few micrometers (μm) [Zipfel et al., “Nonlinear magic: multiphoton microscopy in the biosciences”, Nature Biotechnology vol. 21, p. 1369-1377 (2003)], which leads to excellent optical sectioning capabilities. This feature of laser scanning microscopy allows for the acquisition of multiple in-focus images of thin sections located at selected depths within a sample, the combination of which enabling three-dimensional imaging of thick samples.
In laser scanning fluorescence microscopy for biological applications, a laser beam is generally focused by an objective lens to a diffraction-limited spot size inside or on the surface of a biological specimen. Single-photon (e.g. confocal), two-photon or multiphoton induced fluorescence is generated at the diffraction-limited focal volume. Scattered and reflected laser light, as well as fluorescent emission light from the sample, are re-collected by the objective lens and may be separated by beam splitters. The beam splitters are typically configured to selectively transmit or reflect fluorescence emission while attenuating the scattered and reflected laser light. High-sensitivity photodetectors can be used to detect the selectively filtered fluorescence emission and transform the detected light into an electrical signal, which may be recorded by a computer. By raster scanning the fluorescent sample in three dimensions such as, for example, by using a galvanometer-driven x-y scanner and a piezo-objective z-driver, a volumetric image of the sample may be obtained on a pixel-by-pixel basis, wherein the brightness of each pixel corresponds to the relative intensity of detected light emanating from an elementary volume of the sample. Therefore, imaging a sample whose thickness is larger than the depth of field generally involves acquiring a stack of two-dimensional images at different depths, and adding or averaging these images numerically [Burvall, “Axicon imaging by scalar diffraction theory”, PhD thesis, Royal Institute of Technology, Sweden (2004)]. By way of example, FIG. 1 (PRIOR ART) shows an example of a laser-scanning microscope used for two-photon excited fluorescence.
Although confocal and two-photon laser scanning microscopies share many similarities, two-photon absorption has evolved, due in part to the widespread availability of ultrashort and intense laser pulses, into a particularly powerful tool for vital imaging of biological systems. Two-photon laser scanning microscopy has also alleviated some of the drawbacks of confocal microscopy. In particular, two-photon microscopy provides three-dimensional optical sectioning with limited emission of background fluorescence from outside the plane of focus and reduced photobleaching and photodamage. As a result, this technique can yield improved tissue penetration, as compared to confocal microscopy, while also being less phototoxic to live specimens. Moreover, the non-linear nature of the two-photon process provides intrinsic optical sectioning, which is achieved without a confocal pinhole.
Yet, despite the benefits of the optical sectioning capabilities and the increased spatial resolution achievable by two-photon laser scanning microscopy, the point illumination principle used in this technique reduces the acquisition speed for thick or bulk samples, since a stack of images taken at different depths must be acquired and added. In other words, the maximum acquisition speed of an extended depth-of-field image is therefore N times slower than the acquisition of one two-dimensional image at a single depth, where N is the number of two-dimensional images in the stack. Optical sectioning thus leads to a loss of temporal resolution which may not be suitable when investigating dynamic biological processes for which temporal resolution may be more important than axial resolution. For example, when dynamic interactions between neurons tagged with fluorescent markers are studied, the time between activation of two neuronal cells located at different depths within a specimen may need to be observed [Zipfel et al., “Nonlinear magic: multiphoton microscopy in the biosciences”, Nature Biotechnology vol. 21, p. 1369-1377 (2003); König, “Multiphoton microscopy in life sciences”, Journal of Microscopy vol. 200, p. 83-104 (2000)]. Conventional two-photon laser scanning microscope thus requires scanning a sample along several transverse places, each at a different depth within the sample, in order to cover the whole region of interest, thereby significantly increasing acquisition time and reducing temporal resolution.
In this context, various approaches have been proposed to increase the depth of field of laser scanning microscopes. These depth-of-field-extension methods may be classified into four categories, which are considered in greater detail below.
The first method involves focus elongation through added spherical aberration, as described, for example, in the following documents: Burvall et al., “Simple lens axicon”, Applied Optics vol. 43, p. 4838-4844 (2004); and “System and methods for thick specimen imaging using a microscope based tissue sectioning device”, U.S. Pat. Appl. Pub. No. 2009/0091566 to Turney and Sheard. While the spherical aberration method effectively allows for an increase of the depth of field, the resulting focal spot size varies along the propagation axis. Therefore, image resolution is not constant across the thickness of the sample.
The second method involves wavefront coding with a phase mask followed by digital processing, as described, for example, in the following documents: Tucker et al., “Extended depth of field and aberration control for inexpensive digital microscope systems”, Optics Express vol. 4, p. 467-474 (1999); “Extended depth of field optical systems”, U.S. Pat. No. 7,218,448; and “Method, apparatus and system for extending depth of field (DOF) in a short-wavelength microscope using wavefront encoding”, U.S. Pat. Appl. Pub. No. US 2008/0240347 to Bloom. In the wavefront coding approach, the excitation beam is distorted and the images thus acquired are blurry and must be treated by digital processing before obtaining the effective resolution. The advantage of this method relies mainly on its compatibility with wide-field microscopy. However, it cannot be applied to laser scanning microscopy and relies on numerical post-treatment.
The third method involves a rapid variation of the focal length, as described, for example, in the following documents: Olivier et al., “Two-photon microscopy with simultaneous standard and extended depth of field using a tunable acoustic gradient-index lens”, Optics Letters vol. 34, p. 1684-1686 (2009); Smith et al. “Extended depth-of-field microscopy” Proceedings of SPIE vol. 7570, p. 75700S (2010); Botcherby et al., “Real-time extended depth of field microscopy”, Optics Express vol. 16, p. 21843-21848 (2008); “Extended depth of focus microscopy”, Inter. Pat. Appl. Pub. No. WO 2004/075107 to Dresser; and “Apparatus and method for extended depth of field imaging”, U.S. Pat. Appl. No. 2008/0089598 to George and Chi. The focal depth variation technique consists in rapidly changing the focal plane while acquiring each pixel. The depth of field extension is limited by defocus aberration, which degrades image resolution for large variations in depth. Furthermore, in some cases, the acquired images are blurry and must be treated by digital processing before obtaining the effective resolution.
Finally, the fourth method involves the generation of a non-diffracting beam, as described, for example, in the following documents: Dufour et al., “Two-photon excitation fluorescence microscopy with a high depth of field using an axicon”, Applied Optics, vol. 45, p. 9246-9252 (2006); Arimoto, “Imaging properties of axicon in a scanning optical system”, Applied Optics vol. 31, p. 6653-6657 (1992); “Laser scanning optical system using an axicon”, Eur. Pat. No. 0 627 643 to Ichie; “Laser scanning optical system and laser scanning optical apparatus” U.S. Pat. No. 5,583,342 to Ichie; Botcherby et al., “Scanning two photon fluorescence microscopy with extended depth of field”, Optics Communications vol. 268, p. 253-260 (2006); and “High resolution imaging devices with wide field and extended focus”, U.S. Pat. Appl. No. 2011/0205352 to Pavani et al.
Non-diffracting beams, such as Bessel beams and Mathieu beams, are known to retain their transverse profile while propagating, thereby allowing for lateral image resolution to be maintained throughout the thickness of the sample. In laser scanning microscopy, an interesting type of non-diffracting beam is the Bessel beam whose transverse intensity profile follows a zero-order Bessel function of the first kind characterized by an intense central peak with low-intensity side lobes. While the ideal Bessel beam extends indefinitely in the transverse plane, thus preventing any physical realization of such a beam, it may be experimentally generated to a close approximation by adding thereto a Gaussian envelope. This yields a so-called “Bessel-Gauss beam”, which retains most of the non-diffractive nature of the central peak of the ideal Bessel beam.
Methods have been presented for producing Bessel-Gauss beams as a way to increase the depth of field in laser scanning microscopy and may be classified depending on the type of optical elements, for example refractive or diffractive, employed for their generation.
On the one hand, an axicon can be a conical lens, which is the simplest refractive optical element capable of generating Bessel-Gauss beams [McLeod, “The axicon: a new type of optical element”, Journal of the Optical Society of America, vol. 44, p. 592-597 (1954)]. In 2006, Dufour et al. [Dufour et al., “Two-photon excitation fluorescence microscopy with a high depth of field using an axicon”, Applied Optics, vol. 45, p. 9246-9252 (2006)] proposed to replace the objective lens of a two-photon laser scanning microscope by a large-angle conical lens so as to illuminate the sample with Bessel-Gauss beams having an extended depth of field. However, limitations of this approach include a slow scan rate arising from the need to displace the axicon mechanically, an absence of a proper working distance, a less-than-optimal fluorescence collection, and difficulties in fabricating defect-free large-angle conical lenses and in adjusting the depth of field.
Arimoto et al. [Arimoto, “Imaging properties of axicon in a scanning optical system”, Applied Optics vol. 31, p. 6653-6657 (1992)] describe a laser scanning optical system that incorporates a conical lens to provide an extended depth of focus. The beam-shape characteristics and control of the resulting Bessel beam as well as aberration effects arising from off-axis illumination are experimentally studied. However, scanning the excitation beam in the plane of the sample is considered only along one axis and the system does not include collection and detection of fluorescence. Moreover, while providing a laser scanning microscope based on their design considered, several problems are anticipated, most notably optical aberrations at large scanning angles and low-contrast images due to the side lobes of the Bessel beam.
In another approach, Ichie [“Laser scanning optical system using an axicon”, Eur. Pat. No. 0 627 643 to Ichie; “Laser scanning optical system and laser scanning optical apparatus” U.S. Pat. No. 5,583,342 to Ichie] introduced two identical conical lenses in a laser scanning microscope. The conical lenses are arranged such that apexes thereof are opposed forward or backward to each other. The two conical lenses produce an annular laser beam which then passes through the objective lens of the microscope, thus illuminating the sample with a Bessel beam. However, this system poses severe constraints on the mechanical alignment and the fabrication tolerance error of the conical lenses.
In a further approach, phase-modulating diffractive optical elements have also been proposed in order to increase the depth of field in two-photon laser scanning microscopy. In particular, Botcherby et al. [Botcherby et al., “Scanning two photon fluorescence microscopy with extended depth of field”, Optics Communications vol. 268, p. 253-260 (2006)] used a binary phase-only diffractive optical element to simulate the linear superposition of a positive and a negative axicon and thus convert a laser beam into an annular beam. As in Ichie [“Laser scanning optical system using an axicon”, Eur. Pat. No. 0 627 643 to Ichie; “Laser scanning optical system and laser scanning optical apparatus” U.S. Pat. No. 5,583,342 to Ichie], the annular beam is then converted into a Bessel beam at the sample after passing through the objective lens of the microscope. However, while this system can allow for an increase of the axial extent of the beam illuminating the sample without compromising the lateral resolution, the annular beam thus generated must be spatially filtered to remove higher orders of diffraction. This filtering process leads to a loss of 25% of the optical power and requires two additional lenses. In this regard, it should be emphasized that because Bessel-Gauss beam gradually spread its power as a beam travels along the propagation axis, the intensity of the signal scales inversely to the distance traveled by the beam. For at least this reason, optimizing the power throughput of the microscope and adjusting the depth of field to the thickness of the sample becomes desirable in extended depth-of-field microscopy methods.
In light of the above, a need in the art exists for a system and method capable of providing an extended and adjustable depth of field in laser scanning microscopy with reduced loss of optical power and lateral resolution, while also alleviating at least some of the drawbacks of the prior art.